The long term objective of P.I.'s research is to understand the molecular mechanism(s) underlying the pathogenesis of X-linked retinal diseases. X-linked Retinitis Pigmentosa (XLRP) is a set of progressive retinal degenerations with severe clinical manifestations and visual loss in young adults. The primary cause of photoreceptor degeneration is not known. No treatment is currently possible. The emphasis in this project will be on the identification and characterization of two XLRP genes (RP2 and RP3) that have been mapped in the Xp11.23-p21.1 region. Two complementary strategies of candidate gene identification and positional cloning will be used to accomplish the goal. During the previous R29 grant period, we developed a subtraction- selection strategy to identify candidate retinal or retinal pigment epithelium (RPE) cDNAs from the region of the XLRP disease loci. The human retina, RPE and fetal eye cDNA libraries were constructed and then enriched for tissue-specific genes by an efficient biotin-based subtraction method. We are using a number of strategies for selecting X chromosome-specific cDNAs from the subtracted libraries. Two retinal genes (31A and S3X67) have been identified from the Xp11.23-p21.1 regional. For positional cloning, we have isolated yeast artificial chromosome (YAC) clones from the XLRP region by using several markers. The characterization of YACs has identified novel polymorphic loci and cDNA clones. Studies have also been initiated to perform linkage analysis in Michigan XLRP families. The complimentary approaches of identifying the disease genes by the genomic route of positional cloning and by the cDNA route of isolating candidate retinal and RPE genes will be continued in parallel during the project. Furthermore, novel methods are being developed to select retinal cDNA clones directly from sorted or microdissected X-genomic libraries. The candidate genes will be identified from the Xp11.23-p21.1 region and used to search for specific mutations that may be responsible for the disease. Once XLRP genes are identified, the expression, regulation and function of their products will be studied to understand the molecular mechanisms of the pathogenesis of the disease. The proposed investigations may also uncover candidate genes for other X- linked retinal diseases. The sequence tagged sites, polymorphic markers and cDNA clones, generated during the project, may help in refining the genetic map and constructing the physical map of the human X-chromosome. Pre- and antenatal diagnosis and carrier detection in affected families will be the early benefits of the proposed studies. It is hoped that the better design of therapeutic strategies (including gene therapy) will follow the identification and analysis of the disease gene and its product.